Performance Standards for Antimicrobial Susceptibility testing

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2. Performance Standards for Antimicrobial Susceptibility testing Dr S. Hekmat MD. CAP Reference Laboratory of Iran Dept Of Microbiology AST

3. Antimicrobial Susceptibility Testing( AST ) Indications for Performing AST Methods of AST Reagents for the Disk Difusion Test Procedure for Performing Disk Difusion Test Factors influencing AST General comments and warning Fastidious and problem organisms Quality control in AST • p • General comments and warning AST

4. METHODS OF AST • Diffusion Dilution Diffusion & • Kirby – Bauer method MIC Dilution 1) Broth dilution E – Test method 2) Agar dilution AST

5. Reagents for the Disk Diffusion Test • 1- Mueller Hinton Agar medium • 2- Preparation of Turbidity Standard • 3- Antimicrobial Disks • 4- Storage of antimicrobial discs AST

6. Mueller Hinton Agar Medium • Preparation of Mueller Hinton Agar • PH • Moisture • Effects of Thymidine or Thymine • Effects of Variation in Divalent Cations • Testing Strains That Fail to Grow Satisfactorily AST

7. Turbidity Standard • 0.5 McFarland standard • ( 0.5 ml of 0.048 mol/ml BaCl2 added to • 99.5 ml of 0.18 mol/L H2SO4(1% v/v) • density in 625 nm = 0.08 - 0.1 • Keep in 4-6 ml aliquots in screw cap • tubes in dark , room temperature • Replace or verify density monthly AST

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9. Procedure for Performing DiskDiffusion Test • Inoculum Preparation • Growth Method • Direct Colony Suspension Method • Inoculation of Test Plates • Application of Disks to Inoculated Plates • Reading Plates & Interpreting Results AST

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13. Fastidious or Problem Organisms • Antibiotic susceptibilityof Streptococcus . Pneumoniae • Antibiotic susceptibilityof other Streptococcus spp. • Antibiotic susceptibility ofEnterococcus spp. • Antibiotic susceptibility ofStaphylococcusspp. • Antibiotic susceptibilityof Haemophilus spp. • Antibiotic susceptibilityof Neisseria gonorrhoeae AST

14. Antibiotic susceptibilityof S. pneumonia • Media : Mulller- Hinton with 5 % Sheep blood agar • inoculum : Direct colony suspension in normal saline • Inoculated plates containing disks are incubated at 35C 20–24h and % 5 CO2. • The zone measurement is from the top of plate with the lid removed. • Amoxicillin , Ampicillin , cefepime , cefotaxime, ceftriaxone , imipenem , meropenem may be used to treat pneumococcal infections , but they should be determined by MIC ( not DDM ) • Isolates of S. pneumonia with oxacillin disk zone ≥ 20mm ( MIC < 0.06 ) are susceptible to Penicillin , and can be considered susceptible to : ampicillin , amoxicillin , clavulanic – acid , cephems , imipenem, meropenem and these agents need not to be tested. • Oxacillin zone sizes ≤ 19 mm occur with penicillin – resistant , intermediate strains , so isolates should not be reported as penicillin – resistant or intermediate until Penicillin , ampicillin , cephems , imipenem, meropenem MICs be determind. • Penicillin , cefotaxime or ceftriaxone , and meropenem should be tested by MIC Method and • reported routinely for CSF isolates of S. pneumonia . These isolates should betested for vancomycin also. AST

15. Antibiotic susceptibility of other Streptococcus spp • Medium : Mueller Hinton agar • Inoculum ; Growth or direct method • Incubation ; 35C , ambient air , 16-18 h • Susceptibility testing of penicillins and other β - lactams for treatment of Streptococcus pyogenes or Streptococcus agalactiae is not routinely necessary. • Disk diffusion for penicilin , ampicillin , quinolones are for reporting isolates of beta-hemolytic streptococci only. • Viridans streptococci isolated from blood ,CSF ,.. Should be tested for penicilin , ampicillin susceptibility testing bu MIC method. AST

16. Antibiotic susceptibility ofEnterococcus spp. • Medium ; Mueller- Hinton agar • Inoculum : Direct or growth method • Incubation : 35 c, ambient air , 16-18h ,24h for vancomycin. • For Enterococcus spp. Cephems , aminoglycosides (except for high-level ), clindamycin , SXT , may be active invitro ,but are not clinically effective ,and should not be reported susceptible. • Penicillin susceptibility may be used to predict susceptibility to other penicillins , for non - β - lactamase producing enterococci. • A β - lactamase test is recommended for blood and CSF isolates. A positive test predicts resistance to penicillin as well as amino ,carboxy – and ureidopenicillins. • Enterococcal endocarditis requires combined therapy. AST

17. Antibiotic susceptibility ofStaphylococcus spp. • Medium : Mueller – Hinton agar • Inoculum : Direct colony suspension • Incubation : 35C ambient air , 16-18h , 24h for oxacillin , methicillin , nafcillin ,and vancomycin. • Penicillin- susceptiblestaphare also susceptible to other penicilins, carbapenems, cephems. • Penicillin- resistant , oxacillin – susceptiblestrains are also resistant to β -lactamase –labile penicillins ,but susceptible to other β -lactamase stable penicillins, β -lactamase inhibitors , cephems , carbapenems. • Oxacillin – resistantare resistant to all β -lactam antibiotics. • Susceptibility or resistance to β -lactam antibiotics may be deduced from testing only penicillin and oxacillin ,routine testing of others is not advised. AST

18. Penicillin- resistant , oxacillin – susceptible strains staphs produce β - lactamase and testing of 10-unit penicillin disk instead of ampicillin is preferred. • Penicillin should be tested for all of β - lactamase –labile penicillins. • From β - lactamase- stable penicillins oxacillin is the choice , because is more resistant to degradation in storage, is able to detect heteroresistant strains. Cloxacillin disks should not beused, because they may not detect oxacillin – resistant strains. • For intermediate results perform oxacilin – salt agar test. • Routine testing of urine isolates of S. saprophyticus is not advised if there is not complicated UTI. They usually respond to concentrations achieved in urine by commonly used antibiotics (F/M, SXT, Quinolone,.. ) • MRS are often resistant to multiple classes of antibiotics , like : Aminoglycosides , Quinolones , clindamycin, SXT, macrolides, tetracycline ,.... AST

19. Antibiotic susceptibility of Haemophilus spp. • Medium: Haemophilus Test Medium ( HTM ) • Muller- Hinton agar • 15 µ g/ml β -NAD • 15 µ g/ml bovine hematin • 5 µ 9/ml yeast extract • Inoculum : Direct colony suspension • Incubation ; 35 C , % 5 CO2 , 16 – 18 h. • Only results of ampicilin , one of 3rd generation cephems ,chloramphenicol , meropenem should be reported routinely for CSF isolates of Haemophilus influenzae. • Β – lactamase test can provide a rapid means for detecting ampicilin,amoxicillin resistance. AST

20. Antibiotic susceptibility of Neisseria gonorrhoeae • Medium : GC agar • Inoculum ; Direct colony suspension • Incubation ; 35 C , 5% CO2 , 20-24h • Disk diffusion tests with ampicillin , penicillin , rifampin are unreliable for Nisseria meningitidis , MIC is necessary. • Positive β - lactamase test predicts resistance to ampicillin , penicillin ,amoxicillin. • Gonococci with 10-unit penicillin disk zone ≤ 19 mm are likely to be • β - lactamase producing strains. • β - lactamase test remains preferable to other susceptibility methods for rapid , accurate penicillin resistance. AST

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22. Quality Control Procedures • Purpose • Precision & Accuracy of AST procedure • Performance of used reagents • Performance of responsible personel • Reference Strains for QC • To control Precision & Accuracy AST

23. Enterococcus Faecalis Escherichia Coli Escherichia Coli Staphylococcus aureus Strep. Pneumoniae P. aeruginosa Kleb.Pneumonia Haem. influenzae Stock Strains for Quality Control • ATCC 29212 • ATCC 25922 • ATCC 35218 • ATCC 25923 • ATCC 49619 • ATCC 27853 • ATCC 700603 • ATCC 49247 AST

24. Quality Control Procedures • Storing Quality Control strains • Zone Diameter Quality Control Limits • Frequency of Quality Control Testing • Daily Testing • Weekly ,monthly Testing AST

25. Corrective Action • Out-Of Control Result due to an obvious error • Use of the wrong disk • Use of wrong control strain • Obvious contamination of strain • Wrong procedure AST

26. THANK YOU AST

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